NMR studies of the structure and dynamics of staphylococcal nuclease (SNase) were carried in order to elucidate the catalytic function of this model enzyme. We studied (1) the molecular dynamics of wild type SNase and (2) the three dimensional (3D) structure of an SNase mutant. (1) SNase dynamics. Novel pulse sequences were developed that permitted the accurate measurement of relaxation parameters of heteronuclei (15N and 13C) in proteins. The relaxation parameters of the 22 leucine methyl carbons in SNase were measured. An analysis of the relaxation data showed significant internal motion of nearly one half of the leucine sidechains in SNase liganded to Ca2+ and pdTp. In the absence of the ligands, internal motions increased substantially. These results were interesting because all leucine sidechains are buried, suggesting a dynamic environment in portions of the protein interior. (2) Mutant structure. The SNase mutant, G50F/V51N ~SNase, is considerably more stable than the wild type enzyme, but is 100-500 fold less active. We have shown that the conformations of the mutant and wild type proteins are essentially identical except for a few residues near the active site. The difference in stability is due to the replacement of a disordered loop of the wild type enzyme with a well structured tight turn in the mutant. Several thousand NOE's have been measured for the mutant protein, and work is currently underway to determine its full 3D structure. A comparison of the wild type and mutant structures will be made in order to elucidate the reason for low level of catalytic activity of the mutant protein. The significance of the project lies in the information about (1) the range and time scales of protein structural fluctuations provided by dynamics studies and (2) the relationship between structure and function that comes from comparing the structures of wild type and mutant proteins. In addition, many novel NMR experiments have been developed using SNase as a model system.